Proteins expressed by mycobacterium tuberculosis and not by BCG and their use as diagnostic reagents and vaccines

ABSTRACT

The invention provides polypeptides encoded by open reading frames present in the genome of  Mycobacterium tuberculosis  but absent from the genome of BCG and diagnostic and prophylactic methodologies using these polypeptides.

The invention is in the field of tuberculosis and, specifically,reagents useful for generating immune responses to Mycobacteriumtuberculosis and for diagnosing infection and disease in a subject thathas been exposed to M. tuberculosis.

BACKGROUND OF THE INVENTION

Tuberculosis infection continues to be a world-wide health problem. Thissituation has recently been greatly exacerbated by the emergence ofmulti-drug resistant strains of M. tuberculosis and the internationalAIDS epidemic. It has thus become increasingly important that effectivevaccines against and reliable diagnostic reagents for M. tuberculosis beproduced.

This application is a U.S. national stage entry of internationalapplication number PCT/US00/12257 filed May 4, 2000, which claims thebenefit of U.S. provisional application No. 60/132,505, the disclosuresof which are each incorporated herein by reference in their entireties.The disclosure of U.S. Pat. No. 6,087,163 is incorporated herein byreference in its entirety.

SUMMARY OF THE INVENTION

The invention is based on the inventor's discovery that a polypeptideencoded by an open reading frame (ORF) in the genome of M. tuberculosisthat is absent from the genome of the Bacille Calmette Guerin (BCG)strain of M. bovis elicited a delayed-type hypersensitivity response inanimals infected with M. tuberculosis but not in animals sensitized withBCG. Thus proteins encoded by ORFs present in the genome of M.tuberculosis but absent from the genome of BCG represent reagents thatare useful in discriminating between M. tuberculosis and BCG and, inparticular, for diagnostic methods (e.g., skin tests and in vitro assaysfor M. tuberculosis-specific antibodies and lymphocyte responsiveness)which discriminate between exposure of a subject to M. tuberculosis andvaccination with BCG. The invention features these polypeptides,functional segments thereof, DNA molecules encoding either thepolypeptides or the functional segments, vectors containing the DNAmolecules, cells transformed by the vectors, compositions containing oneor more of any of the above polypeptides, functional segments, or DNAmolecules, and a variety of diagnostic, therapeutic, and prophylactic(vaccine) methodologies utilizing the foregoing.

Specifically, the invention features an isolated DNA molecule containinga DNA sequence encoding a polypeptide with a first amino acid sequencethat can be the amino acid sequence of the polypeptide MTBN1, MTBN2,MTBN3, MTBN4, MTBN5, MTBN6, MTBN7 or MTBN8, as depicted in FIG. 1, or asecond amino acid sequence identical to the first amino acid sequencewith conservative substitutions; the polypeptide has Mycobacteriumtuberculosis specific antigenic and immunogenic properties. Alsoincluded in the invention is an isolated portion of the above DNAmolecule. The portion of the DNA molecule encodes a segment of thepolypeptide shorter than the full-length polypeptide, and the segmenthas Mycobacterium tuberculosis specific antigenic and immunogenicproperties. Other embodiments of the invention are vectors containingthe above DNA molecules and transcriptional and translational regulatorysequences operationally linked to the DNA sequence; the regulatorysequences allow for expression of the polypeptide or functional segmentencoded by the DNA sequence in a cell. The invention encompasses cells(e.g., eukaryotic and prokaryotic cells) transformed with the abovevectors.

The invention encompasses compositions containing any of the abovevectors and a pharmaceutically acceptable diluent or filler. Othercompositions (to be used, for example, as DNA vaccines) can contain atleast two (e.g., three, four, five, six, seven, eight, nine, ten,twelve, fifteen, or twenty) DNA sequences, each encoding a polypeptideof the Mycobacterium tuberculosis complex or a functional segmentthereof, with the DNA sequences being operationally linked totranscriptional and translational regulatory sequences which allow forexpression of each of the polypeptides in a cell of a vertebrate. Insuch compositions, at least one (e.g., two, three, four, five, six,seven, or eight) of the DNA sequences is one of the above DNA moleculesof the invention. The encoded polypeptides will preferably be those notencoded by the genome of cells of the BCG strain of M. bovis.

The invention also features an isolated polypeptide with a first aminoacid sequence that can be the sequence of the polypeptide MTBN1, MTBN2,MTBN3, MTBN4, MTBN5, MTBN6, MTBN7 or MTBN8 as depicted in FIG. 1, or asecond amino acid sequence identical to the first amino acid sequencewith conservative substitutions. The polypeptide has Mycobacteriumtuberculosis specific antigenic and immunogenic properties. Alsoincluded in the invention is an isolated segment of this polypeptide,the segment being shorter than the full-length polypeptide and havingMycobacterium tuberculosis specific antigenic and immunogenicproperties. Other embodiments are compositions containing thepolypeptide, or functional segment, and a pharmaceutically acceptablediluent or filler. Compositions of the invention can also contain atleast two (e.g., three, four, five, six, seven, eight, nine, ten,twelve, fifteen, or twenty) polypeptides of the Mycobacteriumtuberculosis complex, or functional segments thereof, with at least oneof the at least two (e.g., two, three, four, five, six, seven, or eight)polypeptides having the sequence of one of the above describedpolypeptides of the invention. The polypeptides will preferably be thosenot encoded by the genome of cells of the BCG strain of M. bovis.

The invention also features methods of diagnosis. One embodiment is amethod involving: (a) administration of one of the above polypeptidecompositions to a subject suspected of having or being susceptible toMycobacterium tuberculosis infection; and (b) detecting an immuneresponse in the subject to the composition, as an indication that thesubject has or is susceptible to Mycobacterium tuberculosis infection.An example of such a method is a skin test in which the test substance(e.g., compositions containing one or more of MTBN1-MTBN8) is injectedintradermally into the subject and in which a skin delayed-typehypersensitivity response is tested for. Another embodiment is a methodthat involves: (a) providing a population of cells containing CD4 Tlymphocytes from a subject; (b) providing a population of cellscontaining antigen presenting cells (APC) expressing a majorhistocompatibility complex (MHC) class II molecule expressed by thesubject; (c) contacting the CD4 lymphocytes of (a) with the APC of (b)in the presence of one or more of the polypeptides, functional segments,and or polypeptide compositions of the invention; and (d) determiningthe ability of the CD4 lymphocytes to respond to the polypeptide, as anindication that the subject has or is susceptible to Mycobacteriumtuberculosis infection. Another diagnostic method of the inventioninvolves: (a) contacting a polypeptide, a functional segment, or apolypeptide/functional segment composition of the invention with abodily fluid of a subject;

(b) detecting the presence of binding of antibody to the polypeptide,functional segment, or polypeptide/functional segment composition, as anindication that the subject has or is susceptible to Mycobacteriumtuberculosis infection.

Also encompassed by the invention are methods of vaccination. Thesemethods involve administration of any of the above polypeptides,functional segments, or DNA compositions to a subject. The compositionscan be administered alone or with one or more of the other compositions.

As used herein, an “isolated DNA molecule” is a DNA which is one or bothof: not immediately contiguous with one or both of the coding sequenceswith which it is immediately contiguous (i.e., one at the 5′ end and oneat the 3′ end) in the naturally-occurring genome of the organism fromwhich the DNA is derived; or which is substantially free of DNA sequencewith which it occurs in the organism from which the DNA is derived. Theterm includes, for example, a recombinant DNA which incorporated into avector, e.g., into an autonomously replicating plasmid or virus, or intothe genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic fragment produced by PCR orrestriction endonuclease treatment) independent of other DNA sequences.Isolated DNA also includes a recombinant DNA which is part of a hybridDNA encoding additional M. tuberculosis polypeptide sequences.

“DNA molecules” include cDNA, genomic DNA, and synthetic (e.g.,chemically synthesized) DNA. Where single-stranded, the DNA molecule maybe a sense strand or an antisense strand.

An “isolated polypeptide” of the invention is a polypeptide which eitherhas no naturally-occurring counterpart, or has been separated orpurified from components which naturally accompany it, e.g., in M.tuberculosis bacteria. Typically, the polypeptide is considered“isolated” when it is at least 70%, by dry weight, free from theproteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, a preparation of a polypeptide of theinvention is at least 80%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, the peptide of the invention.Since a polypeptide that is chemically synthesized is, by its nature,separated from the components that naturally accompany it, the syntheticpolypeptide is “isolated.”

An isolated polypeptide of the invention can be obtained, for example,by extraction from a natural source (e.g., M. tuberculosis bacteria); byexpression of a recombinant nucleic acid encoding the polypeptide; or bychemical synthesis. A polypeptide that is produced in a cellular systemdifferent from the source from which it naturally originates is“isolated,” because it will be separated from components which naturallyaccompany it. The extent of isolation or purity can be measured by anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

The polypeptides may contain a primary amino acid sequence that has beenmodified from those disclosed herein. Preferably these modificationsconsist of conservative amino acid substitutions. Conservativesubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine, and leucine; asparticacid and glutamic acid; asparagine and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine.

The terms “protein” and “polypeptide” are used herein to describe anychain of amino acids, regardless of length or post-translationalmodification (for example, glycosylation or phosphorylation). Thus, theterm “Mycobacterium tuberculosis polypeptide” includes full-length,naturally occurring Mycobacterium tuberculosis protein, as well arecombinantly or synthetically produced polypeptide that corresponds toa full-length naturally occurring Mycobacterium tuberculosis protein orto particular domains or portions of a naturally occurring protein. Theterm also encompasses a mature Mycobacterium tuberculosis polypeptidewhich has an added amino-terminal methionine (useful for expression inprokaryotic cells) or any short amino acid sequences useful for proteinpurification by affinity chromatography, e.g., polyhistidine forpurification by metal chelate chromatography.

As used herein, “immunogenic” means capable of activating a primary ormemory immune response. Immune responses include responses of CD4+ andCD8+T lymphocytes and B-lymphocytes. In the case of T lymphocytes, suchresponses can be proliferative, and/or cytokine (e.g., interleukin(IL)-2, IL-3, IL-4, IL-5, IL-6, IL-12, IL-13, IL-15, tumor necrosisfactor-α (TNF-α), or interferon-γ (IFN-γ))-producing, or they can resultin generation of cytotoxic T-lymphocytes (CTL). B-lymphocyte responsescan be those resulting in antibody production by the responding Blymphocytes.

As used herein, “antigenic” means capable of being recognized by eitherantibody molecules or antigen-specific T cell receptors (TCR) onactivated effector T cells (e.g., cytokine-producing T cells or CTL).

Thus, polypeptides that have “Mycobacterium tuberculosis specificantigenic properties” are polypeptides that: (a) can be recognized byand bind to antibodies elicited in response to Mycobacteriumtuberculosis organisms or wild-type Mycobacterium tuberculosis molecules(e.g., polypeptides); or (b) contain subsequences which, subsequent toprocessing of the polypeptide by appropriate antigen presenting cells(APC) and bound to appropriate major histocompatibility complex (MHC)molecules, are recognized by and bind to TCR on effector T cellselicited in response to Mycobacterium tuberculosis organisms orwild-type Mycobacterium tuberculosis molecules (e.g., polypeptides).

As used herein, polypeptides that have “Mycobacterium tuberculosisspecific immunogenic properties” are polypeptides that: (a) can elicitthe production of antibodies that recognize and bind to Mycobacteriumtuberculosis organisms or wild-type Mycobacterium tuberculosis molecules(e.g., polypeptides); or (b) contain subsequences which, subsequent toprocessing of the polypeptide by appropriate antigen presenting cells(APC) and bound to appropriate major histocompatibility complex (MHC)molecules on the surface of the APC, activate T cells with TCR thatrecognize and bind to peptide fragments derived by processing by APC ofMycobacterium tuberculosis organisms or wild-type Mycobacteriumtuberculosis molecules (e.g., polypeptides) and bound to MHC moleculeson the surface of the APC. The immune responses elicited in response tothe immunogenic polypeptides are preferably protective. As used herein,“protective” means preventing establishment of an infection or onset ofa disease or lessening the severity of a disease existing in a subject.“Preventing” can include delaying onset, as well as partially orcompletely blocking progress of the disease.

As used herein, a “functional segment of a Mycobacterium tuberculosispolypeptide” is a segment of the polypeptide that has Mycobacteriumtuberculosis specific antigenic and immunogenic properties.

Where a polypeptide, functional segment of a polypeptide, or a mixtureof polypeptides and/or functional segments have been administered (e.g.,by intradermal injection) to a subject for the purpose of testing for aM. tuberculosis infection or susceptibility to such an infection,“detecting an immune response” means examining the subject for signs ofa immunological reaction to the administered material, e.g., reddeningor swelling of the skin at the site of an intradermal injection. Wherethe subject has antibodies to the administered material, the responsewill generally be rapid, e.g., 1 minute to 24 hours. On the other hand,a memory or activated T cell reaction of pre-immunized T lymphocytes inthe subject is generally slower, appearing only after 24 hours and beingmaximal at 24-96 hours.

As used herein, a “subject” can be a human subject or a non-human mammalsuch as a non-human primate, a horse, a bovine animal, a pig, a sheep, agoat, a dog, a cat, a rabbit, a guinea pig, a hamster, a rat, or amouse.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. Unless otherwiseindicated, these materials and methods are illustrative only and are notintended to be limiting. All publications, patent applications, patentsand other references mentioned herein are illustrative only and notintended to be limiting.

Other features and advantages of the invention, e.g., methods ofdiagnosing M. tuberculosis infection, will be apparent from thefollowing description, from the drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a depiction of the amino acid sequences of M.tuberculosis polypeptides MTBN1-MTBN8 (SEQ ID NOs:1-8, respectively).

FIGS. 2A-2E are a depiction of the nucleotide sequences of the codingregions (mtbn1-mtbn8) encoding MTBN1-MTBN8 (SEQ ID NOs:9-16,respectively)

FIG. 3 is a bar graph showing the delayed-type hypersensitivityresponses induced by intradermal injection of 3 different test reagentsin female guinea pigs that had been either infected with M. tuberculosiscells or sensitized with BCG or M. avium cells.

DETAILED DESCRIPTION

The genome of M. tuberculosis [Cole et al. (1998) Nature 393:537-544]contains open reading frames (ORFs) that have been deleted from theavirulent BCG strain. The polypeptides encoded by these ORFs aredesignated herein “M. tuberculosis BCG Negative” polypeptides (“MTBN”)and the ORFs are designated “mtbn.” The invention is based on thediscovery that a MTBN polypeptide (MTBN4) elicited a skin response inanimals infected with M. tuberculosis, but not in animals sensitized toeither BCG or M. avium, a non-M. tuberculosis-complex strain ofmycobacteria (see Example 1 below). These findings indicate that MTBN(e.g., MTBN1-MTBN8) can be used in diagnostic tests that discriminateinfection of a subject by M. tuberculosis from exposure to bothmycobacteria other than the M. tuberculosis-complex and BCG. The M.tuberculosis-complex includes M. tuberculosis, M. bovis, M. microti, andM. africanum. Thus they can be used to discriminate subjects exposed toM. tuberculosis, and thus potentially having or being in danger ofhaving tuberculosis, from subjects that have been vaccinated with BCG,the most widely used tuberculosis vaccine. Diagnostic assays that arecapable of such discrimination represent a major advance that willgreatly reduce wasted effort and consequent costs resulting from furtherdiagnostic tests and/or therapeutic procedures in subjects that havegiven positive results in less discriminatory diagnostic tests.Furthermore, the results in Example 1 show that MTBN4, as expressed bywhole viable M. tuberculosis organisms, is capable of inducing a strongimmune response in subjects infected with the organisms and thus has thepotential to be a vaccine.

The MTBN polypeptides of the invention include, for example,polypeptides encoded within the RD1, RD2, and RD3 regions of the M.tuberculosis genome [Mahairas et al. (1996) J. Bacteriol.178:1274-1282]. Of particular interest are polypeptides encoded by ORFswithin the RD1 region of the M. tuberculosis genome. However, theinvention is not restricted to the RD1, RD2, and RD3 region encodedpolypeptides and includes any polypeptides encoded by ORFs contained inthe genome of one or more members of the M. tuberculosis genome and notcontained in the genome of BCG. The amino acid sequences of MTBN1-MTBN8are shown in FIG. 1 and the nucleotide sequences of mtbn1-mtbn8 areshown in FIG. 2.

The invention encompasses: (a) isolated DNA molecules containing mtbnsequences (e.g., mtbn1-mtbn8) encoding MTBN polypeptides (e.g.,MTBN1-MTBN8) and isolated portions of such DNA molecules that encodepolypeptide segments having antigenic and immunogenic properties (i.e.,functional segments); (b) the MTBN polypeptides themselves (e.g.,MTBN1-MTBN8) and functional segments of them; (c) antibodies (includingantigen binding fragments, e.g., F(ab′)₂, Fab, Fv, and single chain Fvfragments of such antibodies) that bind to the MTBN polypeptides (e.g.,MTBN1-MTBN8) and functional segments; (d) nucleic acid molecules (e.g.,vectors) containing and capable of expressing one or more of the mtbn(e.g., mtbn1-mtbn8) sequences and portions of DNA molecules; (e) cells(e.g., bacterial, yeast, insect, or mammalian cells) transformed by suchvectors;

(f) compositions containing vectors encoding one or more M. tuberculosispolypeptides (or functional segments) including both the MTBN (e.g.,MTBN1-MTBN8) polypeptides (or functional segments thereof) andpreviously described M. tuberculosis polypeptides such as ESAT-6, 14 kDaantigen, MPT63, 19 kDa antigen, MPT64, MPT51, MTC28, 38 kDa antigen,45/47 kDa antigen, MPB70, Ag85 complex, MPT53, and KatG (see also U.S.application Ser. No. 08/796,792); (g) compositions containing one ormore M. tuberculosis polypeptides (or functional segments), includingboth the polypeptides of the invention and previously described M.tuberculosis polypeptides such as those described above; (h)compositions containing one or more of the antibodies described in (c);(i) methods of diagnosis involving either (1) administration (e.g.,intradermal injection) of any of the above polypeptide compositions to asubject suspected of having or being susceptible to M. tuberculosisinfection, (2) in vitro testing of lymphocytes (B-lymphocytes, CD4 Tlymphocytes, and CD8 T lymphocytes) from such a subject forresponsiveness (e.g., by measuring cell proliferation, antibodyproduction, cytokine production, or CTL activity) to any of the abovepolypeptide compositions,(3) testing of a bodily fluid (e.g., blood, saliva, plasma, serum,urine, or semen or a lavage such as a bronchoalveolar lavage, a vaginallavage, or lower gastrointestinal lavage) for antibodies to the MTBNpolypeptides (e.g., MTBN1-MTBN8) or functional segments thereof, or theabove-described polypeptide compositions; (4) testing of a bodily fluid(e.g., as above) for the presence of M. tuberculosis, MTBN (e.g.,MTBN1-MTBN8) polypeptides or functional segments thereof, or theabove-described polypeptide compositions in assays using the antibodiesdescribed in (c); and (5) testing of a tissue (e.g., lung or bronchialtissue) or a body fluid (e.g., as above) for the presence of nucleicacid molecules (e.g., DNA or RNA) encoding MTBN polypeptides (e.g.,MTBN1-MTBN8) (or portions of such a nucleic acid molecules) usingnucleic acid probes or primers having nucleotide sequences of thenucleic molecules, portions of the nucleic molecules, or the complementsof such molecules; and (j) methods of vaccination involvingadministration to a subject of the compositions of either (f), (g), (h)or a combination of any two or even all 3 compositions.

With respect to diagnosis, purified MTBN proteins, functional segmentsof such proteins, or mixtures of proteins and/or the functionalfragments have the above-described advantages of discriminatinginfection by M. tuberculosis from either infection by other bacteria,and in particular, non-pathogenic mycobacteria, or from exposure (by,for example, vaccination) to BCG.

Furthermore, compositions containing the proteins, functional segmentsof the proteins, or mixtures of the proteins and/or the functionalsegments allows for improved quality control since “batch-to-batch”variability is greatly reduced in comparison to complex mixtures such aspurified protein derivative (PPD) of tuberculin.

The use of the above-described polypeptide and nucleic acid reagents forvaccination also provides for highly specific and effectiveimmunization. Since the virulent M. tuberculosis polypeptides encoded bygenes absent from avirulent BCG are likely to be mediators of virulence,immunity directed to them can be especially potent in terms ofprotective capacity. Where vaccination is performed with nucleic acidsboth in vivo and ex vivo methods can be used. In vivo methods involveadministration of the nucleic acids themselves to the subject and exvivo methods involve obtaining cells (e.g., bone marrow cells orfibroblasts) from the subject, transducing the cells with the nucleicacids, preferably selecting or enriching for successfully transducedcells, and administering the transduced cells to the subject.Alternatively, the cells that are transduced and administered to thesubject can be derived from another subject. Methods of vaccination anddiagnosis are described in greater detail in U.S. Pat. No. 6,087,163,the disclosure of which is incorporated herein by reference in itsentirety.

The following example is meant to illustrate, not limit the invention.

EXAMPLE 1 MPBN4 Elicits a Specific Skin Reaction in Guinea Pigs Infectedwith M. tuberculosis

Four groups of outbred female guinea pigs (18 per group) were used totest the usefulness of the MTBN4 polypeptide as a M.tuberculosis-specific diagnostic reagent. The four groups were treatedas follows.

Group 1 animals were infected by aerosol with approximately 100 M.tuberculosis strain H37Rv cells.

Group 2 animals were sensitized intradermally with 10⁶ live M. bovis BCGJapanese cells.

Group 3 animals were sensitized intradermally with 10⁶ live M. aviumcells.

Group 4 animals were mock-sensitized by intradermal injection withsaline.

Seven weeks after infection or sensitization, the animals were injectedintradermally with 1 μg of PPD (6 animals from each group), 2 μg ofpurified recombinant MPT64 (6 animals from each group), or 2 μg of MTBN4(6 animals from each group). The diameter of the resulting erythema wasmeasured 24 hours later. Data are expressed as mean diameter of erythema(in mm) and standard deviations are indicated (FIG. 3).

No erythema was detected in the group 4 animals with any test substanceand thus no data are shown for this group. On the other hand, group 1animals (solid bars) showed a significant response with all three testsubstances. Group 2 animals (open bars) showed a significant response toPPD and MPT64 but not MTBN4. Group 3 animals showed a significantresponse to PPD only (hatched bars).

Thus, PPD which contains antigenic/immunogenic molecules common to theM. tuberculosis-complex as well as other mycobacterial strains, gave theleast discriminatory results in that it induced responses in animalsinfected with or sensitized to mycobacteria of the M.tuberculosis-complex (M. tuberculosis and BCG) as well as anothernon-pathogenic mycobacterium (M. avium). While MPT64, which is encodedand expressed by both M. tuberculosis and BCG, did not elicit a responsein animals infected with M. avium, it did elicit responses in both theM. tuberculosis infected and the BCG sensitized animals. Finally, MTBN4elicited a response in only the M. tuberculosis animals. Thus it inducedthe most specific response and, most importantly, allowed fordiscrimination between animals infected with M. tuberculosis and thosesensitized to BCG.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A vector comprising: (a) a DNA sequence encoding a full length MTBN4polypeptide, wherein the polypeptide is not encoded by the genome of theBacille Calmette Guerin (BCG) strain of Mycobacterium bovis; (b) atleast one additional DNA sequence encoding a polypeptide which isencoded by Mycobacterium tuberculosis but is not encoded by the genomeof the Bacille Calmette Guerin (BCG) strain of Mycobacterium bovis; and(c) each DNA sequence being operationally linked to a regulatorysequence allowing expression of the polypeptide encoded by each DNAsequence in a cell.
 2. A vector comprising: (a) a DNA sequence encodinga segment of a full length MTBN4 polypeptide, wherein said segmentretains an antigenic property of the polypeptide and wherein the segmentis not encoded by the genome of the Bacille Calmette Guerin (BCG) strainof Mycobacterium bovis; (b) at least one additional DNA sequenceencoding a segment of a full length polypeptide which is encoded byMycobacterium tuberculosis but is not encoded by the genome of theBacille Calmette Guerin (BCG) strain of Mycobacterium bovis; and (c)each DNA sequence being operationally linked to a regulatory sequenceallowing expression of the segment encoded by each DNA sequence in acell.
 3. A cell transformed with the vector of claim
 1. 4. A celltransformed with the vector of claim
 2. 5. A composition comprising avector and a pharmaceutically acceptable diluent or filler, wherein thevector comprises a DNA sequence encoding a full length MTBN4 polypeptideand wherein the polypeptide is not encoded by the genome of the BacilleCalmette Guerin (BCG) strain of Mycobacterium bovis and at least oneadditional DNA sequence encoding a polypeptide which is encoded byMycobacterium tuberculosis but is not encoded by the genome of theBacille Calmette Guerin (BCG) strain of Mycobacterium bovis.
 6. Acomposition comprising: at least two DNA sequences, each encoding apolypeptide of the Mycobacterium tuberculosis complex that is not apolypeptide encoded by the genome of the Bacille Calmette Guerin (BCG)strain of Mycobacterium bovis, and each being operationally linked to aregulatory sequence which allows expression of said polypeptide in acell of a vertebrate, wherein at least one of said DNA sequences encodesa full length MTBN4 polypeptide.
 7. A composition comprising: at leasttwo DNA sequences, each encoding a polypeptide of the Mycobacteriumtuberculosis complex that is not a polypeptide encoded by the genome ofthe Bacille Calmette Guerin (BCG) strain of Mycobacterium bovis, andeach being operationally linked to a regulatory sequence which allowsexpression of said polypeptide in a cell of a vertebrate, wherein atleast one of said at least two DNA sequences encodes a segment of a fulllength MTBN4 polypeptide, wherein said segment retains an antigenicproperty of the polypeptide.